Blood coagulation is a process consisting of a complex interact ion of various blood components (or factors) that eventually gives rise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascade,” are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as “active factors” and are designated by the addition of the letter “a” to the name of the coagulation factor (e.g., Factor VIIa).
Initiation of the haemostatic process is mediated by the formation of a complex between tissue factor, which is exposed to the circulating blood following injury to the vessel wall, and Factor VIIa, which is present in the circulation in an amount corresponding to about 1% of the total Factor VII protein mass. This complex is anchored to the tissue factor-bearing cell and converts Factors IX and X to their active forms Factor IXa and Factor Xa on the cell surface. Factor Xa converts prothrombin to thrombin on the tissue factor-bearing cell, which activates Factor VIII, Factor V, Factor XI, and Factor XIII. Furthermore, the limited amount of thrombin formed in this initial step of haemostasis also activates the platelets. Following the action of thrombin on the platelets, the platelets change shape and expose charged phospholipids on their surface. This activated platelet surface forms the template for further Factor X activation and the full thrombin generation. The further Factor X activation on the activated platelet surface occurs via a Factor IXa and Factor VIIIa complex formed on the surface of the activated platelet, and Factor Xa then converts prothrombin into thrombin while still on the surface. Thrombin then converts fibrinogen into fibrin, which is insoluble and which stabilizes the initial platelet plug. This process is localized to the site of the tissue factor exposure thereby minimizing the risk of a systemic activation of the coagulation system. In recent years, Factor VII and tissue factor have been found to be the main initiators of blood coagulation.
Factor VIIa is produced from its precursor, Factor VII, which is synthesized in the liver and secreted into the blood where it circulates as a single-chain glycoprotein (molecular weight of about 50,000 Da). Wild-type Factor VII as used herein has the amino acid sequence and nucleotide sequence disclosed in FIGS. 1 and 2. The term “Factor VII” is meant to encompass Factor VII polypeptides in their uncleaved form (the zymogen form) as well as those that have been proteolytically or otherwise processed to yield their respective bioactive forms, which may be referred to as Factor VIIa. Wild type Factor VII is cleaved typically between residues 152 and 153 to produce Factor VIIa.
Factor VII is converted in vitro into the two-chain form Factor VIIa by Factor Xa, Factor XIIa, Factor IXa, or thrombin. Like several other plasma proteins involved in haemostasis, Factor VII is dependent on Vitamin K for its activity, which is required for the gamma-carboxylation of multiple glutamic acid residues that are clustered close to the amino terminus of the protein. These gamma-carboxylated glutamic acids are required for the metal ion-induced interaction of Factor VII with phospholipids. In the presence of tissue factor, phospholipids, and calcium ions, the two-chain Factor VIIa rapidly activates Factor X or Factor IX by limited proteolysis. Factor VIIa is susceptible to proteolytic cleavage, giving rise to a number of degradation products that do not have clotting activity.
Factor VII variants having an amino acid sequence derived from wild type Factor VII by substitution, deletion, and/or insertion of one or more amino acids have been published. For example, Dickinson et al. (Proc. Natl. Acad. Sci USA (1996) 93, 14379-14384) relates to Factor VII variants wherein Lys157, Val158, Glu296, Met298, Asp334, Ser336, or Lys227 have been individually replaced by Ala. Iwanaga et al. (Thromb. Haemost. (supplement August 1999), 466, abstract 1474) relates to Factor VIIa variants wherein residues 316-320 are deleted or residues 311-322 are replaced with the corresponding residues from trypsin. U.S. Pat. App. Pub. 2008/0058255 A1 to Bolt et al. relates to Factor VII variants having a glycosylation-disrupting substitution at either N145 or N322, or at both N145 and N322. Toso et al. reported a series of Factor VII structure-function studies based on naturally occurring mutations. The mutant recombinant Factor VII proteins included T324M, E385K, and two mutant Factor VII proteins lacking glycosylation core sequences in either the Factor VII heavy chain (N322Q) or the Factor VII light chain (N145Q). Toso et al., “Lack of Heavy Chain Glycosylation in Patient with Factor VII Deficiency Not Responsible for Mutant FVIIa Activity,” Blood, vol. 96, no. 11, part 2 (16 Nov. 2000), p. 79b (42nd Annual Meeting of the American Society of Hematology).
Most naturally occurring peptides and proteins contain carbohydrate moieties attached to the peptide or protein via specific linkages to a select number of amino acids along the length of the primary peptide or protein chain. Thus, many naturally occurring peptides and proteins are termed “glycopeptides” or “glycoproteins,” respectively. The variability of the glycosylation pattern on any given peptide or protein can impact the function of that peptide or protein. For example, the structure of the N-linked glycans on a peptide or protein can impact various characteristics of the peptide or protein, including the protease susceptibility, intracellular trafficking, secretion, tissue targeting, biological half-life, and antigenicity of the peptide or protein in a cell or organism. The alteration of one or more of these characteristics can affect the efficacy of a peptide or protein in its natural setting, and can also affect the efficacy of the peptide or protein as a therapeutic agent in situations where the peptide or protein has been generated for that purpose.
The carbohydrate structure attached to the peptide or protein chain is known as a “glycan” molecule. The specific glycan structure present on a peptide or protein affects the solubility and aggregation characteristics of the peptide or protein, the folding of the primary peptide or protein chain, and, therefore, its functional or enzymatic activity, the resistance of the peptide or protein to proteolytic attack, and the control of proteolysis leading to the conversion of inactive forms of the peptide or protein to active forms. For example, terminal sialic acid residues present on the glycan molecule affect the length of the half-life of the peptide or protein in the mammalian circulatory system. Peptides and proteins whose glycans do not contain terminal sialic acid residues generally are more rapidly removed from the circulation by the liver.
The glycan structures found in naturally occurring glycopeptides and glycoproteins are typically divided into two classes, N-linked and O-linked glycans. Wild type Factor VIIa contains two N-linked and two O-linked glycosylation sites. N-linked glycosylation is the most common covalent modification in eukaryotes. N-linked glycosylation occurs at the consensus sequence Asn-X-Ser/Thr, where the glycan attaches to the amine group of asparagine and X represents any amino acid except proline. N-linked glycans are based on the common core pentasaccharide, Man3(GlcNAc)2, which can be further modified by the addition of monosaccharides such as N-acetyl galactosamine, galactose, neuraminic acid, N-acetylglucosamine, fructose, mannose, and fucose. The Man3(GlcNAc)2 core with various monosaccharides including terminal sialic acids may be attached via a N-acetylglucosamine to at the Asn in the Asn-X-Ser/Thr consensus sequence. This chemically complex co-translational modification serves many purposes and affects the biology of the protein in diverse ways including proper folding, functional group orientation, and clearance rates.
A variety of methods have been proposed in the art to customize the glycosylation pattern of a peptide or protein, including those described in U.S. Pat. No. 8,008,252 to DeFrees et al.
It is often desirable to stimulate or improve the coagulation cascade in a subject. Factor VIIa has been used to control bleeding disorders caused by clotting factor deficiencies (e.g., haemophilia A and B or deficiency of coagulation Factors XI or VII) or clotting factor inhibitors. Recombinant Factor VIIa, manufactured and sold by Novo Nordisk under the trade name NovoSeven®, is approved for the for the treatment of bleeding episodes in hemophilia A or B patients with inhibitors to Factor VIII or Factor IX and in patients with acquired hemophilia; prevention of bleeding in surgical interventions or invasive procedures in hemophilia A or B patients with inhibitors to Factor VIII or Factor IX and in patients with acquired hemophilia; treatment of bleeding episodes in patients with congenital Factor VII deficiency and prevention of bleeding in surgical interventions or invasive procedures in patients with congenital Factor VII deficiency. U.S. Pat. No. 5,180,583 to Hedner discloses using Factor VIIa to control excessive bleeding in situations not caused by clotting factor defects or clotting factor inhibitors. Hedner discloses treating bleeding disorders caused for example by a defective platelet function, thrombocytopenia, or von Willebrand's disease, and compositions for those uses.
There is a need to treat bleeding from disorders not caused by congenital or developed clotting factor deficiencies or inhibitors to clotting factors. Several clinical trials have demonstrated the efficacy of recombinant Factor VIIa to control bleeds. However, there are concerns over an increase in undesirable thromboembolic events from use of this molecule. Bleeding is a major problem in many disorders, such as in connection with surgery, complications following surgery, stem and organ transplants, intracranial hemorrhage, aortic aneurysm, and trauma, or overdose of certain anti-coagulants.